Project Summary/Abstract The molecular chaperone and prolyl isomerase Pin1 is a promising target for cancer therapy. Knockout of PIN1 yields only mild degenerative phenotypes in normal mice, but greatly impairs tumorigenesis in oncogenic mouse models and decreases proliferation in prostate and breast cancer xenograft models. The reliance of cancer cells on Pin1 activity seems to occur through its ability to stabilize multiple oncogenes and destabilize multiple tumor suppressors. However, despite Pin1's great potential as a therapeutic target, no chemical inhibitor which is both specific and biologically-active has been reported. The challenge is that Pin1 has the features of a classically ?undruggable? protein; it has a shallow, polar binding pocket which has, thus far, prevented discovery of viable inhibitors. We propose a previously-unexplored option for imparting permeability on existing potent in vitro inhibitors of Pin1 as well as expand this SAR using disulfide fragment tethering. A key innovation of this study is that we have designed and preliminarily tested the first potent and cell permeable inhibitors of Pin1 using high affinity phosphate-baring compounds and imparting permeability through phosphoramidate caging. These permeate the lipid bilayer and are subsequently enzymatically liberated to a phosphate-baring active metabolite. In exciting preliminary results, our top compound (DS-2(R)) has single-digit nanomolar EC50 for cyclin D1 destabilization in vivo ? a known Pin1 inhibition biomarker. Herein, I propose (SA 1) rigorous physical and functional characterization of the biological activity of the first potent and cell-active Pin1 inhibitors. Since we are yet unsure of the specificity of these molecules, to supplement this approach, we propose (SA 2) the characterization and improvement of the specificity of our preliminary lead compound DS-2(R). This approach arose from observations that Pin1 is able to tolerate bulky substitutions in place of the benzothiophene which would likely lead to reduced affinity for off-targets. Thus, in collaboration with pioneering experts at UCSF (Jim Wells, Michelle Arkin), we will screen a disulfide-containing fragment library for pharmacophores which engage the bulk-tolerant hydrophobic shelf to prepare analogues which will maintain potent interaction with Pin1 while hopefully reducing ligand complementarity with off- targets. We will use the information gained in the disulfide tethering screen to synthesize and evaluate a directed series of DS-2(R) analogues for improved Pin1 specificity. Overall, the goal of this project is to leverage modern design principles to characterize and improve the first biologically active Pin1 chemical probes. These molecules will facilitate consistent and quality research of Pin1 in oncology and potentially lead to new insights into its suitability as a target. Critically, the proposed studies will greatly expand my training as a chemical biologist by combining structure-based design, modern screening methods, and compound and target validation methods.